ES2633732T3 - Procedure and kit to determine in vitro the immune status of an individual - Google Patents

Abstract

Procedure to determine in vitro the overall immune status of a septic individual for the implementation of the best possible therapy based on the established immune response according to which: a. a blood sample of the individual is available, b. There are at least two specific reagents of at least two expression products of at least two target genes respectively selected from the following target genes: HLA-DRA, S100A9, S100A8, IRAK-M, LY64, CIITA, TNF-alpha, IL -10, GBP1, MMP7, CXCL1, CXCL10, COX2, FCN1, TNFAIP6, CXCL7, CXCL5, PID1 and RHOU, c. the expression of said at least two target genes is determined, and d. the expression of said at least two target genes respectively is compared with a reference expression, a modification in the expression of said at least two target genes with respect to their reference expression being an indicator of a modification of the individual's immune status, and being a correlation between the expression of said at least two target genes with their reference expression an indicator of a normality of the individual's immune status.

Description

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By hybridization, the process is understood during which, under appropriate conditions, two nucleotide fragments, such as for example a hybridization probe and a target nucleotide fragment, which have sufficiently complementary sequences are capable of forming a double strand with hydrogen bonds Stable and specific. A nucleotide fragment "capable of hybridizing" with a polynucleotide is a fragment that can hybridize with said polynucleotide under hybridization conditions that can be determined in each case in a known manner. The hybridization conditions are determined by the rigor, that is to say the rigor of the conditions of realization. Hybridization is even more specific as it is performed more rigorously. The stringency is defined in particular as a function of the composition based on a probe / target duplex, as well as the degree of mismatch between two nucleic acids. The rigor may also depend on the parameters of the reaction, such as the concentration and type of ionic species present in the hybridization solution, the nature and concentration of denaturing agents and / or the temperature of hybridization. The stringency of the conditions in which a hybridization reaction must be performed will depend mainly on the hybridization probes used. All these data are of course known and the appropriate conditions can be determined by the person skilled in the art. In general, depending on the length of the hybridization probes used, the temperature for the hybridization reaction is between about 20 and 70 ° C, in particular between 35 and 65 ° C in a saline solution at a concentration of about 0.5 to 1 M. A hybridization reaction detection stage is then performed.

By detection, it is understood either a direct detection by a physical method, or a detection method with the help of a marker. Numerous methods of detection exist for the detection of nucleic acids [1,2].

A marker means a plotter capable of generating a signal. A non-limiting list of these tracers comprises enzymes that produce a detectable signal, for example by colyrometry, fluorescence or luminescence, such as horseradish peroxidase, alkaline phosphatase, -galactosidase, glucose-6-phosphate dehydrogenase; chromophores such as fluorescent, luminescent or dye compounds; electron density groups detectable by electron microscopy or by their electrical properties such as conductivity, by amperometry or voltammetry methods, or by impedance measurements; the groups detectable by optical methods such as diffraction, surface plasmon resonance, contact angle variation or by physical methods such as atomic force spectroscopy, tunnel effect, etc .; radioactive molecules such as 32P, 35S or 125I. Thus, the polynucleotide can be labeled during the enzymatic amplification step, for example using a labeled triphosphate nucleotide for the amplification reaction. The labeled nucleotide will be a deoxyribonucleotide in amplification systems that generate a DNA, such as PCR, or a ribonucleotide in amplification techniques that generate an RNA, such as TMA or NASBA techniques. The polynucleotide can also be labeled after the amplification step, for example by hybridizing a labeled probe according to the sandwich hybridization technique described in WO-A-91/19812.

In the sense of the present invention, the hybridization probe may be a so-called capture probe. In this case, the target nucleotide fragment can be previously labeled by a marker. The so-called capture probe is immobilized or can be immobilized on a solid support by any appropriate means, that is directly or indirectly, for example by covalence or adsorption. A hybridization reaction is then performed between said detection probe and the labeled target nucleotide fragment.

The hybridization probe may also be a so-called detection probe. In this case, the hybridization probe can be labeled by a marker. A hybridization reaction is then performed between said capture probe and the target nucleotide fragment.

Whether a so-called capture probe or a so-called detection probe is used, the hybridization reaction can be performed on a solid support that includes all the materials on which a nucleic acid can be immobilized. As solid support, synthetic materials or natural materials, possibly chemically modified, can be used, in particular polysaccharides such as cellulose-based materials, for example paper, cellulose derivatives such as cellulose acetate and nitrocellulose or dextran, polymers, copolymers, in particular based on styrene monomers, natural fibers such as cotton, and synthetic fibers such as nylon; mineral materials such as silica, quartz, glass, ceramics; latex; Magnetic particles; metal derivatives, gels, etc. The solid support may be in the form of a microtiter plate, of a membrane as described in application WO-A-94/12670, of a particle or of a biochip.

In the present invention, the determination of the expression of a target gene can be analyzed by the expression of mRNAs that are transcribed at a given time. In this case, the biological material is a nucleic acid, and the specific reagent can be indifferently an amplification primer or a hybridization probe as defined above.

The expression of a target gene can be determined as follows:

1) After extracting the total RNAs from a blood sample or PBMCs, a reverse transcription step is performed, as described above in order to obtain the different complementary DNAs of the different

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Messenger RNAs initially present in the blood sample or in the PBMCs (or cDNA),

2) cDNAs are specifically amplified. In this case, the specific reagent used comprises at least one amplification primer specific to the target gene, as defined above. This step can be carried out in particular by a PCR amplification reaction or by any other appropriate amplification technique,

3) expression of the target gene is determined by quantifying the cDNAs. The cDNAs can be quantified in particular by the use of a quantification interval obtained by an amplification reaction carried out until saturation. In order to take into account the variability of enzymatic efficacy that can be observed during different stages (reverse transcription, quantitative PCR, etc.), the expression of the target gene of the different groups of patients can be normalized, by simultaneously determining the expression of a gene called domestic, whose expression is similar in different groups of patients. By making a relationship between the expression of the target gene and the expression of the domestic gene, any variability between the different experiments is corrected. The person skilled in the art may refer in particular to the following publications: [3-4] Bustin SA Journal of molecular endocrinology, 2002, 29: 23-39; Giulietti A Methods, 2001, 25: 386-401.

The expression of a target gene can also be determined as follows:

1) after extracting the total RNAs from a blood sample or from PBMCs, a reverse transcription step is performed, as described above, in order to obtain the different complementary DNAs of the different messenger RNAs initially present in the sample or PBMCs (or cDNA)

2) cDNAs are immobilized on a membrane

3) the expression of the target gene is determined by hybridizing the cDNAs to a previously labeled target gene hybridization probes. Such hybridization techniques are well known to those skilled in the art, and the Northern transfer technique can be cited in particular. This hybridization reaction can be performed after a specific amplification step of the complementary DNAs of the messenger RNAs of a target gene when in particular the gene is weakly expressed.

The expression of a target gene can also be performed by the expression of the proteins encoded by the target gene. In this case, the biological material is a protein and several detection techniques can be used with or without ligand. Mass spectrometry can be used as a detection technique without ligand. The specific reagent may be a protein specific antibody encoded by the target gene for a ligand detection system.

Recombinant antibodies, specific to the translated protein of the target gene can be obtained according to classical procedures known to those skilled in the art, from prokaryotic organisms, such as bacteria, or from eukaryotic organisms, such as yeasts, mammalian cells, of plants, of insects

or of animals, or through extra-cellular production systems.

Monoclonal antibodies can be prepared according to the classical techniques known to those skilled in the art such as the hybridoma technique whose general principle is recalled below.

Initially, an animal, usually a mouse, (or cells in culture in the field of in vitro immunizations) is immunized with a target antigen of interest, whose B lymphocytes are then capable of producing antibodies against said antigen. These antibody producing lymphocytes are then fused with "immortal" myelomatous cells (murine in the example) to give rise to hybridomas. From the heterogeneous mixture of the cells thus obtained, a selection is then made of the cells capable of producing a particular antibody and multiplying indefinitely. Each hybridoma is multiplied in the form of a clone, each leading to the production of a monoclonal antibody whose recognition properties against the antigen of interest may be tested for example in ELISA, by immunoblotting in one or two dimensions, in immunofluorescence, or with the Help from a biosensor. The monoclonal antibodies so selected are then purified in particular according to the affinity chromatography technique.

Antibody fragments can be obtained for example by proteolysis. Thus, they can be obtained by enzymatic digestion, resulting in Fab type fragments (treatment with papain [5] or type F (ab) '2 (treatment with pepsin; [6]. They can also be prepared recombinantly [7] Another antibody fragment that is suitable for the purposes of the invention comprises an Fv fragment which is a dimer constituted of the non-covalent association of the light variable domain (VL) and the heavy variable domain (VH) of the Fab fragment, hence the association of two polypeptide chains In order to improve the stability of the Fv fragment due to the separation of two polypeptide chains, this Fv fragment can be engineered by inserting an adapted peptide bond between the VL domain and the VH domain [8]. There is talk of a scFv fragment ("single chain Fragment variable") since it is made up of a single polypeptide chain, the use of a peptide bond preferably composed of 15 to 25 amino acids allows to join the C-terminal end of a

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presence or not of immunostimulants. The determination was made by an ELISA test on culture supernatants obtained after 45 hours. The axis of the ordinates represents the protein concentrations (pg / ml) of TNF- (Figure 4A) and IL-10 (Figure 4B). Black histograms represent cells stimulated twice by LPS without prodrug (control). The white histograms correspond to cells stimulated twice by LPS in the presence of IFN-b1b (Imukin ™, Boehringer, Ingelheim, Austria) at 100 ng / ml, the gray histograms in the presence of 100 ng / ml of GM-CSF ( Sigma-Aldrich, Deisenhofen, Alemnia) and the histograms with stripes in the presence of 100 ng / ml of FLT3-L (Sigma-Aldrich, Deisenhofen, Germany). The data are represented on average +/- standard deviation. A test with Wicoxon apparatus was performed for the statistical analysis of the results, correcting for the test number performed: * means that p <0.017 against cells stimulated twice with LPS without prodrug.

Figure 6: Figure 6 shows the expression of TNF alpha (A), IL-10 (B), HLA-DRA (C), CIITA (D), IRAK-M (E), ABIN-3 (F ), LY64 (G), S100A9 (H), S100A8 (I), GBP1 (J), MMP7 (K), CXCL1 (L), CXCL10 (M), COX2 (N), FCN1 (O), TNFAIP6 (P ), CXCL7 (Q), CXCL5 (R), PID1 (S) and RHOU (T) by PBMCs, from 7 healthy volunteers, stimulated twice by LPS (2 ng / ml after 100 ng / ml) in the presence or not of prodrugs, and quantified by biochip after 45 hours. The axis of the ordinates represents the fluorescence intensity of each of the hybridization probes: (A) 207113_s_at for TNFa; (B) 207433_at for IL-10; (C) 208894_at for HLA-DRA; (D) 205101_at for CIITA; (E) 213817_at for IRAK-M; (F) 220655_at for ABIN-3; (G) 206206_at for LY64; (H) 203535_at for S100A9; (I) 202917_s_at for S100A8; (J) 202269_x_at for GBP1; (K) 204259_art for MMP7; (L) 204470_at for CXCL1; (M) 204533_at for CXCL10; (N) 204748_at for COX2; (O) 205237_at for FCN1; (P) 206026_s_at for TNFAIP6; (Q) 214146_s_at for CXCL7; (R) 215101_s_at for CXCL5; (S) 219093_at for PID1 and (T) 223168_at for RHOU. Black histograms represent cells stimulated twice by LPS without prodrug (control). The white histograms correspond to cells stimulated twice by LPS in the presence of IFN-b1b at 100 ng / ml, the gray histograms in the presence of 100 ng / ml of GM-CSF and the histograms with stripes in the presence of 100 ng / ml of FLT3-L. The data are represented on average +/- standard deviation. A t-test was carried out with an apparatus for the statistical analysis of the results, correcting for the test number carried out: * means that p <0.05 against cells stimulated twice with LPS without immunostimulant.

Figure 7: Figure 7 shows the expression of the TNFa (A), IL-10 (B), HLA-DRA (C), CIITA (D), IRAK-M (E), ABIN-3 (F) genes , LY64 (G), S100A9 (H), S100A8 (I), GBP1 (J), MMP7 (K), CXCL1 (L), CXCL10 (M), COX2 (N), FCN1 (O), TNFAIP6 (P) , CXCL7 (Q), CXCL5 (R), PID1 (S) and RHOU (T) by PBMCs, from 7 healthy volunteers, stimulated twice by LPS (2 ng / ml after 100 ng / ml) in the presence or absence of prodrugs, and quantified by qRT-PCR after 45 hours. The axis of the ordinates represents the percentages of induction of expression of the genes mentioned above. Black histograms represent cells stimulated twice by LPS without prodrug (control). The white histograms correspond to cells stimulated twice by LPS in the presence of IFN-b1b at 100 ng / ml, the gray histograms in the presence of 100 ng / ml of GM-CSF and the histograms with stripes in the presence of 100 ng / ml of FLT3-L. The data are represented on average +/- standard deviation. A Wilcoxon apparatus test was performed for the statistical analysis of the results, correcting for the test number performed: * means that p <0.05 against cells stimulated twice with LPS without immunostimulant.

Figure 8: Figures 8A and 8B respectively represent the concentrations of TNF- and IL-10 produced by whole blood cells, from 5 patients in septic shock extracted between D1 and D3, and 5 healthy volunteers, stimulated or not (control) by LPS at 100 ng / ml +/- 100 ng / ml of IFN-1b. Protein determinations have been performed by an ELISA assay on culture supernatants obtained after 15 hours. The axis of the ordinates represents the protein concentrations (pg / ml) of TNF- (Figure 6A) and IL-10 (Figure 6B). Black histograms represent patients in septic shock and blank histograms represent healthy volunteers. The data is represented by means +/- standard deviation.

Figure 9: Figure 9 represents the expression of the TNF-α (A), IL-10 (B), HLA-DRA (C), CIITA (D), GBP1 (E), CXCL10 (F), COX2 genes (G), TNFAIP6 (H) and CXCL5 (I) by whole blood cells, from 5 patients in septic shock extracted between D1 and D3 and 5 healthy volunteers, stimulated or not (controls) by LPS at 100 ng / ml +/- 100 ng / ml of IFN-1b. The quantification of these genes has been carried out by qRT-PCR on the cellular residues obtained after 15 hours. The axis of the ordinates represents the percentages of induction of expression of the genes mentioned above. Black histograms represent patients in septic shock and blank histograms represent healthy volunteers.

Examples

The following examples are given by way of illustration and have no limitation. They allow a better understanding of the invention.

Materials and methods

Preparation of PBMCs and experimental parameters of the endotoxin tolerance (ET) model

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Table

Gen

GenBank accession number Primers SEQ ID NO

IL-10

NM_000572.2 5'-AATAAGGTTTCTCAAGGGGCT-3 ' sense one

5'-AGAACCAAGACCCAGACATCAA-3 '

anti-sense 2

HLA-DRA

NM_019111.4 5'-GCCAACCTGGAAATCATGACA-3 ' sense 3

5'-AGGGCTGTTCGTGAGCACA-3 '

anti-sense 4

APPOINTMENT

NM_000246.3 5'-GCTGGGATTCCTACACAATGC-3 ' sense 5

5'-CGGGTTCTGAGTAGAGCTCAATCT-3 '

anti-sense 6

IRAK-M

NM_007199.2 5'-TTTGAATGCAGCCAGTCTGA-3 ' sense 7

NM_001142523.1

5'-GCATTGCTTATGGAGCCAAT-3 ' anti-sense 8

ABIN-3

NM_024873.4 5'-GAATTCCCAGATAAAAGCTTGT-3 ' sense 9

NM 001128843.1

5'-GACAGTCTGGTGGGTGCTC-3 ' anti-sense 10

S100A9

NM_002965.3 5'-TCAAAGAGCTGGTGCGAAAA-3 ' sense eleven

5'-AACTCCTCGAAGCTCAGCTG-3 '

anti-sense 12

S100AB

NM_002964.4 5'-ATTTCCATGCCGTCTACAGG-3 ' sense 13

5'-CACCAGAATGAGGAACTCCT-3 '

anti-sense 14

LY64

NM_005582.2 5'-GCATTGAGAAAGAAGCCAACAA-3 ' sense fifteen

5'-GAAAAGTGTCTTCATGTATCC-3 '

anti-sense 16

Transcriptional approach to biochip in patients in septic shock

Several blood samples collected directly in PAXgeneTM tubes (PreAnalytiX to Qiagen / BD Company) were obtained from 19 patients in septic shock at the time of shock diagnosis (i.e. within 2 hours after the start of vasopressor treatment) and 48 hours later, as described previously [9]. Total RNA has been extracted with the help of PAXgene Blood RNA kit ™ (PreAnalytix). A step of digestion with DNAse (Qiagen) has been carried out during the extraction in order to eliminate any trace of DNA. The integrity and quality of the RNAs were verified by capillary electrophoresis on the AgilentTM 2100 bioanalyzer (Agilent). The biochips were made according to the manufacturer's instructions (Affymetrix). The reverse transcription reaction of the mRNAs has been performed with the Affymetrix protocol from 3 µg of total RNA. A polyT oligonucleotide primer has been used to determine only mRNAs contained in the solution of total RNAs during reverse transcription. All the stages have been carried out with the Affymetrix -GeneChip® One-Cycle Target Labeling and Control Reagents kit. Reverse transcription products (ANDc) were then used for the synthesis of biotin-labeled cRNA for 16 hours at 37 ° C. After the cRNA purification step to remove unincorporated dNTPs, the cRNAs were quantified and fragmented. Fragmentation control is performed by capillary electrophoresis (Agilent). The cRNA hybridization is performed with the GeneChipHuman Genome U133 Plus 2 biochip before the signal amplification stage by incubation with the SAPE mixture containing Straptavidin Phycoerytrhine after the goat anti-streptavidin IgG antibodies mixed with the biotinylated anti-biotinylated antibody Goat IgG This last stage uses the Fluidic FS450 platform. The analysis of the Affymetrix data begins with the capture of the biochip image by the Affymetrix GeneChip 3000 scanner. The data were then normalized by RMA (Robust Multiple -Array Average) (Irizarry RA, Biostatistics 2003). All data based on the signal strength were used after normalization.

Effect of IFN- in a total blood model of patients in septic shock

The blood of 5 patients in septic shock has been collected in EDTA tube, between D1 and D3 according to the onset of the shock, as well as in 5 healthy volunteers. After centrifugation and plasma extraction, 3 ml of ½ blood have been diluted in 3 ml of RPMI 1640 medium (Eurobio, Courtaboeuf, France) then grown in the presence or absence (control group) of 100 ng / ml of LPS +/- 100 ng / ml of IFN-1b (Imukin) overnight at 37 ° C and 5% CO2 (15 hours). For each condition, supernatants as well as cell debris were recovered and stored at -80 ° C for the ELISA determination of TNF- and IL-10 and at -20 ° C for RNA extraction and quantification by qRT-PCR, as described previously.

Statistical analysis

The results are expressed as mean  standard deviation (SD). The statistical analysis was carried out by the Wilcoxon apparatus test.

Results

ET Model Description

ET models are characterized by a reduction in TNF- production associated with an increase in IL-10 secretion after a second stimulation by LPS. The production of these cytokines was therefore measured

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in the culture supernatant of PBMCs after one or two stimulations by LPS. As shown in Figure 1A, cells stimulated twice by LPS produce low amounts of TNF- (58.5  68.3 pg / ml) compared to cells stimulated only once (1208  594 pg / ml) . On the contrary, as shown in Figure 1B, these same cells secrete stronger concentrations of IL-10 (63.2  58.2 pg / ml) compared to cells stimulated once (8.4  6, 9 pg / ml). In response to a second endotoxin stimulation, cells already stimulated release less TNF- (58.5 ± 68.3 pg / ml) than cells stimulated once (1208 ± 594 pg / ml) and produce more IL-10 (63.2 ± 58.2 versus 8.4 ± 6.9 pg / ml) than cells stimulated once). These results are confirmed at mRNA level since a significant decrease in TNF- expression (Figure 2A) coupled with an increase in IL-10 gene expression (Figure 2B) in cells stimulated twice by LPS has been observed. compared to stimulated PBMCs only once.

An analysis of the gene expression of PBMCs subjected to one or two stimulations with LPS has been performed in Affymetrix biochip. Genes whose expression differs more between one or two stimulations with LPS (p <0.05 and difference in expression> 2) as well as genes known in the literature for being involved in this refractory state of ET or the immunosuppression phase induced by Severe septic states are represented in Figure 2. The second bacterial endotoxin stimulation is accompanied by an increase in mRNA expression of IL-10 (B), HLA-DRA (C), CIITA (D), IRAK-M (E), ABIN-3 (F) while the expression of TNF alpha (A) and LY64 (G) is diminished. Gene expression of alarms S100A9 (H) and S100A8 (I), of chemokines CXCL1 (L), CXCL5 (R), et CXCL7 (Q), of MMP7 (K), FCN1 (O), PID1 (S) and RHOU (T) also increase after a second stimulation with LPS. Finally, the expression of interferon-induced genes GBP1 (J) and CXCL10 (M) as well as TNFAIP6 (P) decreases.

These data have been confirmed later by qRT-PCR (Figure 3) in which the same profile as that obtained on the biochips has been observed. In response to a second endotoxin challenge, the inability to produce and express TNF- combined with the increased production and expression of IL-10, of cells that have previously been exposed to a first dose of LPS, clearly indicates the development of a state of ET.

Analysis of gene expression in patients in septic shock

In order to illustrate the clinical relevance of the model, the expression of these genes in patients in septic shock has been studied retrospectively, at the time of shock diagnosis and 48 hours later. Gene expression has been evaluated by a biochip approach in a cohort of 19 patients described previously [9] and 9 healthy volunteers. As observed in the ET model, the results show an increase in the expression of IL10, IRAK-M, CXCL7 and RHOU (Figures 4A, B, M and O), coupled with a decrease in the expression of Ly64, GBP1 and CXCL10 (Figures 4 F, G and I) in patients in septic shock at the time of diagnosis and even 48 hours later compared to healthy volunteers. In parallel, a slight decrease in TNF-expresión expression is observed in patients (Figure 4E).

Taken together, the clinical data confirm the mRNA modifications observed ex vivo in the ET model and underline the clinical relevance of this model.

IFN- and GM-CSF significantly improve the production of TNF- in PBMCs deactivated by LPS

As indicated in Figure 1, PBMCs stimulated for the first time with LPS show a marked decrease in their production capacity in TNF- in response to a second exposure to endotoxin. Next, it has been investigated, in 7 successive experiments, if a 24-hour incubation in the presence of different immunomodulatory prodrugs (IFN-, GM-CSF and Flt3-L) could restore this synthesis in TNF- and decrease that of IL -10. IFN- and GM-GSF achieve this goal by inducing an increase in TNF- production (Figure 5A). It is interesting to note that the prodrugs tested have an impact only on the reinforcement of the pro-inflammatory side, but remain without effect on the decrease in IL-10 production (Figure 5B).

MRNA monitoring of the effect of immunostimulatory prodrugs during ET

Next, in these same experiments that show a beneficial effect of IFN- and GM-CSF on the restoration of TNF-síntesis synthesis in response to a second stimulation with LPS, the expression modulation of a panel of genes is has studied by biochip (figure 6) then validated by qRT-PCR (figure 7).

A significant induction of TNF- mRNA expression is observed in the presence of IFN- compared to the control values (Figure 6A). The addition of prodrugs induces a slight decrease in mRNA expression of IL10 (Figure 6B). In parallel, a significant increase in gene expression of HLA-DRA and CIITA is observed when cells pretreated with LPS are incubated with IFN- (Figure 6C, 6D). As for the negative regulators of the LPS signaling pathway, ABIN-3 expression decreases significantly in the presence of IFN- (Figure 6E, 6F). The expression of the LY64 and IRAKM genes also decreases in the presence of IFN- (Figure 5G). The three prodrugs decrease the mRNA expression of alarms S100A9 and S100A8 (Figure 6H, 6I). In addition, the presence of IFN-significativamente significantly increases the expression of the GBP1 (J), CXCL10 (M), COX 2 (N) genes,

Claims (1)

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